This proposal will test the hypothesis that downregulation of the signal transducer and activator of transcription 3 (STAT3) will optimize the efficacy of breast cancer immunotherapy with a minigene vaccine targeting tumor cells and immune cells in the tumor microenvironment (TME). The rationale for this approach is based on a large body of evidence indicating that the STAT3 transcription factor inhibits the expression of mediators necessary for immune activation against tumor cells and propagates crosstalk between tumor cells and immune cells in the TME leading to marked tumor-induced immunosuppression. The three specific aims are designed to validate STAT3 as an effective target to provide the necessary synergy between the TME and a minigene vaccine targeting Legumain to achieve optimal immunotherapy of metastatic breast cancer. First, proof of concept will be established for a small probe STAT3 inhibitor (NSC295558) to effectively downregulate STAT3 signaling in tumor-associated macrophages (TAMs) and murine 4T1 breast cancer cells once this inhibitor is incorporated into liposomal nanoparticles (NPs) and specifically targeted and delivered to these cell types. Specific targeting is achieved by conjugating the aminophospholipid of NPs with an azapeptide epoxide inhibitor of Legumain, an asparaginyl endopeptidase specifically overexpressed by TAMs and 4T1 tumor cells in the TME. Targeted and loaded NPs will then be tested for their ability to selectively inhibit STAT3 transcriptional activity in vitro and to induce apoptosis of 4T1 cells and TAMs harboring constitutively activated STAT3. Second, proof of principle will be established that this strategy is valid in downregulating IL-6/JAK/STAT3 signaling in vivo in our syngeneic, spontaneous 4T1 breast cancer metastasis model in BALB/c mice. Emphasis will be on assessing whether STAT3 downregulation correlates with downregulation of T-regulatory (TReg) cells and with genes encoding IL-6 and TGF-[unreadable] that are indicators of tumor induced immunosuppression. Correlation of STAT3 downregulation will also be assessed with increased expression of proinflammatory cytokines and chemokines in TAMs as well as with the ablation of immunosuppressive factors in 4T1 tumor cells. Third, to establish proof of concept that downregulation of STAT3 creates the type of tumor microenvironment with the minimal immunosuppressive activity required to effectively synergize with the anti-tumor effects of our Legumain-based minigene vaccine to induce a long-lived CD8+ memory T cell response which prevents or delays the recurrence of established metastatic breast cancer. Emphasis will be on elucidation of cellular and molecular immunological mechanisms of innate and adaptive immunity responsible for anti-tumor effects of our combination immunotherapy. PUBLIC HEALTH RELEVANCE: The relevance of this research to public health is to establish proof of concept in a mouse model system that specific downregulation of the STAT3 transcription factor decreases the production of immunosuppressive factors and thereby creates a favorable immunological tumor microenvironment which effectively synergizes with a Legumain- based minigene vaccine to significantly improve immunotherapy of established metastatic breast cancer.